Chemistry Reference
In-Depth Information
while pol II and pol III are active in the nucleoplasm. During transcription by those
enzymes, RNAs arematured and released fromthe sites. They thenmove towards the
nuclear envelope to translocate in the cytoplasm through the nuclear pore structure.
Beyond this elementary information, what are the actual RNA dynamics in all these
processes?
8.3.1
Dynamics in Transcription
Most of our knowledge about transcription comes from ensemble measurements
using methods such as the Northern blot, RT-PCR and microarray. These analyses
only provide results that are averages for speci
c molecules in a population and
obscure the differences among all cells. Therefore, only a single cell approach can
provide insight into the dynamic behavior and responses to speci
c stimuli of an
individual cell. Exploiting the MS2 system in bacteria, E. coli transcripts were tracked
and details of prokaryotic transcription revealed [29, 30]. It was demonstrated that
transcription in prokaryotes occurred in bursts with an average of 6min of
activation for approximately 37min of inactivity and that RNA partitioning during
cell division was random, decreasing the correlation between RNA and protein at the
beginning of the cell cycle. Recently, the same approach was used in the eukaryote
Dictyostelium discoideum for the characterization of the transcription of an endoge-
nous developmental gene [31]. Discrete pulses of gene activity were found with an
estimated mean time of 5
-
6min on or off. The length and intensity of the pulse were
consistent during development. This was surprising, considering the strong changes
in transcriptional stimuli occurring throughout differentiation of this organism. The
important conclusion for this developmental system was that the number of pulses
during development did not increase, but rather there was an increase in the number
of cells that became committed to transcribing the gene. Initiation of synchronous
transcription in neighboring cells was observed to be more frequent than predicted
by random events. Furthermore, a transcriptional memory existed in cells that had
already transcribed that gene; they weremore prone to restart transcription than cells
that had never expressed it.
The study of transcriptional dynamics is at its beginning and yet very promising.
Developing of sensitive systems to observe speci
c transcription in real-time
in mammalian cells, will open new frontiers and most likely reveal new insights
into gene expression. A
first step in this direction has been made in the study of
pol II transcription in vivo [24]. Transcription of a speci
c locus was monitored by
FRAP of YFP-pol II recruited to the active site as well as both FRAP and photo-
activation of GFP-MCP labeling the transcribed RNA. A systems-modeling
approach combined with quanti
cation and testing of the model using transcription
inhibitors provided suf
cient resolution to demonstrate a faster transcription rate
coupled with pausing steps during elongation. Variations in the period length and
percentage of pol II pausing could possibly correlate with the appearance of
transcriptional bursting after relief of the block caused by the upstream pausing
polymerase.
